Sleep improving agent

ABSTRACT

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.

TECHNICAL FIELD

The present application is filed claiming the priority of the JapanesePatent Application No. 2016-105477, the entire contents of which areherein incorporated by reference.

The present invention relates to a heat shock proteinexpression-inducing agent, sleep improving agent, anti-stress agent andautonomic nerve-adjusting agent.

BACKGROUND ART

Organisms have various defense mechanisms to adapt to the growthenvironment. For example, when the growth temperature increases by 5-10°C., the three-dimensional structure of cellular proteins starts tochange by thermal denaturation. A group of proteins called “chaperones”is known to involve in the mechanism for repairing this change. Heatshock proteins (hereinafter also referred to as “HSPs”) are known asrepresentative proteins that have a chaperone function. In mammals, morethan ten kinds of HSPs have been currently reported and classified intoHSP 20 family, HSP 40 family, HSP 70 family, HSP 90 family, and the likeon the basis of the molecular weight thereof.

HSPs are also called stress proteins, of which the expression isenhanced in vivo in response to environmental stress, pathologicalstress, psychological stress and the like. HSPs expressed in vivo havethe following role: to bind specifically to proteins that have losttheir original physiological functions because of a partial change ofthe higher-order structure thereof under various stress, and utilizeenergy generated by hydrolysis of ATP to return the higher-orderstructure of the binding partner protein to its original structure, andrestore the physiological function of the protein.

Thus, HSPs contribute to biological defense and maintaining homeostasisof living bodies. If HSP expression can be induced in vivo, it will beeffective in preventing or ameliorating various diseases and symptomscaused by abnormality of higher-order structure of proteins or variousdiseases and symptoms related to HSPs.

Among HSPs, HSP70 has been actively studied in particular. In recentyears, the relationship of HSP70 with sleep (Non-patent Documents 1 and2) or stress (Non-patent Documents 3 and 4) has become clear. It is alsoknown that there is a close relationship between stress and autonomicnervous disorders. Therefore, the chaperone function of HSP 70 isconsidered to contribute to recovery from damages such as sleepdeprivation, daily stress, autonomic neuropathy and the like.

For this reason, HSP70 expression-inducing substances have beenresearched in order to apply them to pharmaceuticals, quasi-drugs, andthe like. As such a substance, for example, Zerumbone, etc. have beenreported (Patent Document 1).

CITATION LIST Patent Documents

-   Patent Document 1: WO 2012/043808

Non-Patent Documents

-   Non-Patent Document 1: Doklady Biological Sciences, 2013, Vol. 449,    pp. 89-92-   Non-Patent Document 2: Doklady Biological Sciences, 2015, Vol. 461,    pp. 76-79-   Non-Patent Document 3: Exp. Ther. Med. 2012, October; 4(4), 627-632-   Non-Patent Document 4: J. Biol. Chem. 2000, Vol. 275, No. 27,    20822-20828

SUMMARY OF INVENTION Technical Problem

The object of the present invention is to provide a HSPexpression-inducing agent, sleep improving agent, anti-stress agent,autonomic nerve-adjusting agent or the like, comprising an activeingredient having an excellent HSP expression inducing activity.

Solution to Problem

As a result of intensive studies to attain the above object, the presentinventors have found that a proline-containing 3-alkyl diketopiperazinerepresented by the following general formula (I) or a salt thereof hasan excellent HSP expression-inducing activity, and based thereon, itshows sleep improving activity, anti-stress activity, autonomicnerve-adjusting activity and the like. The present invention has beenaccomplished on the basis of these findings.

The present invention includes the following embodiments.

[1] A heat shock protein expression-inducing agent comprising a compoundrepresented by formula (I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.[2] The heat shock protein expression-inducing agent according to [1],wherein R is isopropyl, phenyl, or hydroxyphenyl.[3] The heat shock protein expression-inducing agent according to [1],wherein R is isopropyl.[4] The heat shock protein expression-inducing agent according to any of[1]-[3], which is in the form of a pharmaceutical composition,quasi-drug composition or food and beverage composition.[5] A sleep improving agent comprising a compound represented by formula(I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.[6] The sleep improving agent according to [5], wherein R is isopropyl,phenyl, or hydroxyphenyl.[7] The sleep improving agent according to [5], wherein R is isopropyl.[8] The sleep improving agent according to any of [5]-[7], which is inthe form of a pharmaceutical composition, quasi-drug composition or foodand beverage composition.[9] An autonomic nerve-adjusting agent comprising a compound representedby formula (I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.[10] The autonomic nerve-adjusting agent according to [9], wherein R isisopropyl, phenyl, or hydroxyphenyl.[11] The autonomic nerve-adjusting agent according to [9], wherein R isisopropyl.[12] The autonomic nerve-adjusting agent according to any of [9]-[11],which is in the form of a pharmaceutical composition, quasi-drugcomposition or food and beverage composition.[13] An anti-stress agent comprising a compound represented by formula(I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.[14] The anti-stress agent according to claim 13, wherein R isisopropyl, phenyl, or hydroxyphenyl.[15] The anti-stress agent according to claim 13, wherein R isisopropyl.[16] The anti-stress agent according to any of any of [13]-[15], whichis in the form of a pharmaceutical composition, quasi-drug compositionor food and beverage composition.

Effect of Invention

According to the pharmaceutical composition of the present invention, itis possible to provide a HSP expression-inducing agent, sleep improvingagent, anti-stress agent, autonomic nerve-adjusting agent and the like,which has one or more functions or effects described below:

-   (1) inducing the expression of HSP70,-   (2) improving the quality of sleep, specifically one or more    followings: sleep quality, sleep-awakening rhythm, deep sleep    feeling, feeling when waking up, feeling tired when getting up,    sleepiness during the day, work efficiency, gloomy feeling resulting    from unsatisfactory sleep, and the like,-   (3) anti-stress activity,-   (4) autonomic nerve-adjusting activity,-   (5) others, preventing or ameliorating various diseases and symptoms    caused by abnormality of higher-order structure of proteins or    various diseases and symptoms related to HSPs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a figure showing an HSP70 mRNA expression level by aproline-containing 3-alkyl diketopiperazine (Compound 1).

FIG. 2 is a figure showing a HSP70 mRNA expression level by aproline-containing 3-alkyl diketopiperazine (Compound 2).

FIG. 3 is a figure showing a HSP70 mRNA expression level by aproline-containing 3-alkyl diketopiperazine (Compound 3).

FIG. 4 shows the transition of activity level of rats in the lightperiod after the phase advance.

FIG. 5 is a figure showing the activity level of rats on Day 1, lightperiod (12 hours) after the phase advance.

DESCRIPTION OF EMBODIMENTS

The HSP expression-inducing agent according to the present inventioncomprises a proline-containing 3-alkyl diketopiperazine represented bygeneral formula (I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereofas an active ingredient.

The “lower alkyl” in the above formula (I) means a linear or branchedalkyl group having 1-6 carbon atoms. Specific examples thereof includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, neopentyl, hexyl, isohexyl and the like. Preferredis a linear or branched alkyl group having 1-4 carbon atoms such asisopropyl.

In the above formula (I), R is a lower alkyl, phenyl, or hydroxyphenyl,preferably isopropyl, phenyl, or hydroxyphenyl, particularly preferablyisopropyl.

Among the proline-containing 3-alkyl diketopiperazine of the aboveformula (I), particularly preferred are the following Compounds 1-3(particularly, Compound 1).

Compound 1

-   Name: (3S,8aS)-3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione-   Abbreviated name: Cyclo(L-Leu-L-Pro)

Compound 2

-   Name: (3S,8aS)-3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione-   Abbreviated name: Cyclo(L-Phe-L-Pro)

Compound 3

-   Name:    (3S,8aS)-3-(4-hydroxybenzyl)hexahydropyrrolo[1,2-a]pyrazine-1,4-dione-   Abbreviated name: Cyclo(L-Tyr-L-Pro)

Salts of the proline-containing 3-alkyl diketopiperazine include thoseacceptable in the field of pharmaceuticals or foods and beverages, forexample, salts with inorganic bases, salts with organic bases, saltswith basic amino acids, etc.

Suitable examples of salts with inorganic bases include, for example,alkali metal salts such as sodium salt, potassium salt and the like;alkaline earth metal salts such as calcium salt, magnesium salt and thelike; ammonium salt and the like.

Suitable examples of salts with organic bases include, for example,salts with alkylamines (trimethylamine, triethylamine and the like),heterocyclic amines (pyridine, picoline and the like), alkanolamines(ethanolamine, diethanolamine, triethanolamine and the like),dicyclohexylamine, N, N′-dibenzylethylenediamine, or the like.

Preferable examples of salts with basic amino acids include, forexample, salts with arginine, lysine, ornithine, or the like.

Among these salts, alkali metal salts or alkaline earth metal salts arepreferable, and sodium salt is particularly preferable.

The proline-containing 3-alkyl diketopiperazine or a salt thereof usedin the present invention can be prepared according to a method known inthe art, for example, a chemical synthesis method, an enzymatic method,a microorganism fermentation method, or the like. Specifically, thecompound can be synthesized by subjecting a linear peptide todehydration/cyclization reaction, for example, according to a methodshown in JP 2003-252896 A or J. Peptide Sci., 10, 737-737 (2004).

In addition, the above-mentioned Compounds 1-3 are known compounds, andthus can be obtained, for example, from BACHEM.

In the present invention, the above proline-containing 3-alkyldiketopiperazine or a salt thereof may be used alone or in combinationof two or more kinds thereof, as an active ingredient. For example, oneof the Compounds 1-3 may be used alone, or two or three of them may beused together.

For instance, when two of the Compounds 1-3 are used, the usage ratio isnot particularly limited. When two kinds of Compounds 1 and 2, are used,the ratio thereof is, for example, Compound 1:Compound 2=10-0.5:3-0.1,preferably 6-1:2-0.5. When two kinds of Compounds 1 and 3 are used, theratio thereof is, for example, Compound 1:Compound 3=10-0.5:5-0.5,preferably 6-1:4-0.7. When two kinds of Compounds 2 and 3 are used, theratio thereof is, for example, Compound 2:Compound 3=3-0.1:5-0.5,preferably 0.5:4-0.7.

In addition, for instance, when all (three) kinds of the Compounds 1-3are used, the usage ratio is not particularly limited, but include, forexample, Compound 1:Compound 2:Compound 3=10-0.5:3-0.1:5-0.5, preferably6-1:2-0.5:4-0.7.

The proline-containing 3-alkyl diketopiperazine or a salt thereof has anexcellent HSP expression-inducing activity. Therefore, the HSPexpression-inducing agent of the present invention is effective inpreventing or ameliorating various diseases and symptoms caused byabnormality of the higher-order structure of proteins or variousdiseases and symptoms related to HSPs.

In the present invention, HSPs of which the expression is inducedinclude any of HSP20 family proteins such as HSP20, HSP27, and HSP28;HSP40 family proteins such as HSP40 and HSP47; HSP70 family proteinssuch as HSP70, HSP72, and HSP73; HSP90 family proteins such as HSP90aand HSP90b. Preferred are HSP70 family proteins, and more preferred isHSP70.

In the present invention, the induction of expression of HSPs includesexpression in vivo and in vitro, preferably expression in vivo. The invivo site where the expression of HSP is induced is preferably withinthe brain.

The proline-containing 3-alkyl diketopiperazine or a salt thereof showsan excellent sleep improving activity, anti-stress activity, autonomicnerve-adjusting activity, and the like, on the basis of its HSPexpression-inducing activity.

Therefore, the present invention provides a sleep improving agent,anti-stress agent, autonomic nerve-adjusting agent, and the like,comprising the proline-containing 3-alkyl diketopiperazine or a saltthereof as an active ingredient.

The “sleep improving” in the present invention means that improvedeffects on one or more followings: sleep quality, sleep-awakeningrhythm, deep sleep feeling, feeling when waking up, feeling tired whengetting up, sleepiness during the day, work efficiency, gloomy feelingresulting from unsatisfactory sleep, and the like are observed.

The “improving the quality of sleep” means to obtain a good quality ofsleep is obtained by shortening the sleeping latency, decreasing thenumber and time of arousal during sleep, and smoothing the transition toslow-wave sleep which is regard as deep sleep after sleep onset toincrease sleep time.

The “improving sleep-awakening rhythm” means to improve the shift ofsleep-awakening rhythm or to adjust sleeping/waking up rhythm. The shiftof sleep-awakening rhythm also includes social jet lag where social timeand biological clock do not match. The social jet lag is caused by anight-time light environment and a big change of working environment,etc., that is, it means that bedtime/wakeup rhythm shifts on weekdayswith social restrictions such as work and on holidays withoutrestrictions, or the circadian rhythm of the living body collapses dueto irregular sleep cycles and the like.

By improving sleep quality and/or sleep-awakening rhythm, deep sleepfeeling that you can sleep soundly and a refresh feeling when waking upcan be obtained. Furthermore, the work efficiency rises without feelingsleepiness during the day or feeling tired when getting up. Also, it ispossible to reduce sleeping feeling caused by not getting a good qualitysleep or shifting a sleeping/waking up rhythm.

The improvement effect means improvement of symptoms or conditions,prevention or delay of deterioration of symptoms or conditions, orreversal, prevention or delay of the progress of symptoms.

The “anti-stress activity” in the present invention means an activitycapable of preventing fatigue caused by stress in a mammal, particularlyhuman, or preventing fatigue due to stress in the brain. Here, fatigueis a phenomenon in which physical and mental performance temporarilydeclines, when a physical or mental load, is given consecutively. Thedecline of performance is a qualitative or quantitative decline inphysical and mental abilities.

The “autonomic nerve-adjusting activity” in the present invention meansto improve a disturbance in the balance of sympathetic andparasympathetic balance (autonomic nervous balance) caused by excessivestress burden, decreased level of autonomic nervous activity, and thelike. For example, since the function of the gastrointestinal tract ismainly dominated by the parasympathetic sensory nerve, if thesympathetic tension is maintained by a stress load, the action of thegastrointestinal tract is suppressed, thereby causing gastrointestinaldisorders such as anorexia and constipation. In addition, it isconsidered that insomnia is caused when the parasympathetic nerve doesnot function well due to a stress load and the activity of thesympathetic nerve is continued to accelerate. While there is a closerelationship between stress and autonomic nervousness as describedabove, there is also autonomic nervous disorder not due to stress load.Stress load does not necessarily cause autonomic nervous disorders, butmay induce other physical symptoms.

In the present invention, the proline-containing 3-alkyldiketopiperazine or a salt thereof can be used as it is as a HSPexpression-inducing agent, sleep improving agent, anti-stress agent,autonomic nerve-adjusting agent, or the like (hereinafter referred to as“HSP expression-inducing agent or the like”). Also, it can be used incombination with carriers that can be added to pharmaceuticals orquasi-drugs, or food additives, to the extent that they do not affectthe intended function or effect.

In the present invention, the proline-containing 3-alkyldiketopiperazine or a salt thereof can be added to variouspharmaceuticals, quasi-drugs, foods and beverages as a HSPexpression-inducing agent, and thus used in the form of pharmaceuticalcompositions, quasi-drug compositions, food and beverage compositionsand the like.

The pharmaceuticals, quasi-drugs, foods and beverages are notparticularly limited as long as they are usually used, and examplesthereof include tablets, granules, capsules, powders, chewable tablets,confectioneries (cookies, biscuits, chocolate confectionery, chips,cake, gum, candy, gummi, sweet buns, sweet jellied adzuki-bean paste,pudding, jelly, yogurt, ice cream, sherbet and the like), breads,noodles, rice, cereal foods, beverages (liquid, soft drinks, carbonateddrinks, nutritional drinks, powdered drinks, fruit drinks, milk drinks,jelly drinks and the like), soups (powder soups, freeze-dried soups),miso soups (powder miso soups, freeze-dried miso soups) and the like.

In addition, foods and beverages include foods with function claims,foods for specified health uses, health food, nutritional supplements(supplements), foods for medical use and the like.

The subjects to be applied by the HSP expression-inducing agent or thelike in the present invention are not particularly limited, but include,for example, subjects who have subjective dissatisfaction with sleepingfeeling such as shallow asleep, insufficient sleep feeling when gettingup, poor sleep, insufficient deep sleep feeling (cannot sleep deeply),bad dreaming, unrecovered fatigue, and the like; subjects who have nosubjective complaints about sleeping feeling, but want to improve sleepquality because they are feeling fatigue; subjects who feels disorder ofautonomic nerve; and subjects who are stressed in everyday life; and thelike. Also, subjects with no particular problem can take up the agentdaily for the purpose of maintaining good sleep, preventing sleepdisorders, improving sleep quality, autonomic nerve-adjusting activity,anti-stress activity and the like.

The subjects to which the present invention applies are especiallyhumans. The present invention is also applicable to mammals other thanhumans (e.g., mouse, rat, hamster, monkey, cow, horse, pig, sheep, goat,dog, cat, guinea pig, rabbit and the like).

In the HSP expression-inducing agent or the like of the presentinvention, the daily dose (intake) of the proline-containing 3-alkyldiketopiperazine or a salt thereof is not particularly limited and itmay be set as appropriate according to the purpose of administration(target disease and symptom), administration form, sex, weight and ageof subject, etc., but is usually 0.1 μg-1.7 g as a proline-containing3-alkyl diketopiperazine in the case of a human adult, preferably 1μg-1.5 g, and more preferably 5 μg-1.3 g.

In the case of a human adult, the daily dose of Compound 1 is usually0.5 μg-500 mg, preferably 1 μg-450 mg, more preferably 10 μg-400 mg.

In the case of a human adult, the daily dose of: Compound 2 is usually0.1 μg-400 mg, preferably 2 μg-350 mg, more preferably 5 μg-300 mg.

In the case of a human adult, the daily dose of Compound 3 is usually0.1 μg-800 mg, preferably 2 μg-700 mg, more preferably 5 μg-600 mg.

The timing of administration of the HSP expression-inducing agent or thelike of the present invention is not particularly limited, but it ispreferably administered (consumed) once a day before going to bed.

In addition, the present invention provides the proline-containing3-alkyl diketopiperazine or a salt thereof for use as a HSPexpression-inducing agent, sleep improving agent, anti-stress agent,autonomic nerve-adjusting agent, or the like.

Also, the present invention provides use of the proline-containing3-alkyl diketopiperazine or a salt thereof for the preparation of a HSPexpression-inducing agent, sleep improving agent, anti-stress agent,autonomic nerve-adjusting agent, or the like.

Furthermore, the present invention provides a method for inducing HSPexpression, improving sleep, anti-stressing, adjusting autonomic nerve,or the like, which comprises administrating the proline-containing3-alkyl diketopiperazine or a salt thereof to a subject.

In these embodiments, the definitions of the terms, theproline-containing 3-alkyl diketopiperazine or a salt thereof, the formof use, the subject, the dosage, the timing of administration, theblending amount in each agent, etc. are the same as described above.

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to representative examples, but the present invention is notlimited to the following examples. Unless otherwise specified, “%” means“wt %”.

(Example 1) Evaluation of HSP70 mRNA Expression-Inducing Activity

The HSP70 expression-inducing activity by Compounds 1-3 was evaluated bymeasuring a HSP70 mRNA expression level.

Human promyelocytic leukemia cells (HL-60 cells) was suspended in RPMI1640 medium supplemented with 10% fetal bovine serum, and wastransferred into a 1.5 mL tube (45,000 cells/0.9 mL). To the tube wasadded 0.1 mL of each sample that had been prepared in a 1% DMSO solutionand the tube was allowed to stand at 37° C. for 4 hours. To a controlwas added 0.1 mL of 1% DMSO solution. After cultured for 4 hours, thecells was centrifuged by a small refrigerated centrifuge (1,000 g×5minutes) to precipitate the HL-60 cells. After removal of thesupernatant, the cells were lysed in 0.25 mL Trizol (manufactured byThermo Fisher Scientific) and then total RNA was extracted according tothe protocol. The RNA was dissolved in RNase-free water, and theconcentration of the RNA (wave length 260 nm) was measured by using amicrovolume spectrophotometer NanoDrop2000/2000c (manufactured by ThermoFisher Scientific). The RNA solution was diluted with RNase-free waterso as to have a concentration of 50 ng/mL, and then cDNA was synthesizedby using ReverTra Ace® qPCR RT Kit (manufactured by TOYOBO). Thereaction liquid (10 μl) after the reverse transcription was diluted withRNase-free water so as to be 100 μL, and then used as a template forPCR. Beta 2 microglobulin gene was used as an internal control gene forcorrection of the HSP70 gene expression. The primer sequences of genesused for the PCR reaction are as follows.

HSP70 forward primer: (SEQ ID NO: 1) GCATTTCCTAGTATTTCTGTTTGTHSP70 reverse primer: (SEQ ID NO: 2) AATAGTCGTAAGATGGCAGTATABeta 2 microblobulin forward primer: (SEQ ID NO: 3) TAGCTGTGCTCGCGCTACTBeta 2 microblobulin reverse primer: (SEQ ID NO: 4) AGTGGGGGTGAATTCAGTGT

The expression was analyzed by using CFX Connect Real-Time PCR DetectionSystem (manufactured by Bio-rad). 10 μL of the PCR reaction liquid wasincubated at 95° C. for 3 minutes (initial denaturation), followed byrepetition of 55 cycles comprising denaturation at 95° C. for 10seconds, and annealing and extension at 57.8° C. for 30 seconds.Finally, the melting curve was measured every 0.2° C. in the range from65° C. to 95° C. Expression levels of HSP70 gene and internal standardgene were analyzed using Bio-Rad CFX Manager 3.1.

The abilities of inducing HSP70 mRNA expression of Compounds 1, 2, and 3are shown in FIGS. 1-3 by percentages for the control, respectively.From these results, it was revealed that each sample enhanced mRNAexpression of HSP70 gene as compared with the control. In additionconcentration dependence of each active ingredient on HSP70 mRNAexpression inducing ability was investigated. As a result, theconcentrations at which HSP70 mRNA expression inducing ability wasincreased up to 150% relative to the control were 0.083 mg/ml forCompound 1, 0.045 mg/ml for Compound 2, and 0.101 mg/ml for Compound 3.

Example 2

Firstly, 7-week old male Wistar rats were implanted intraperitoneallywith a small activity meter (NanoTaq®, Kissei Comtech Co., Ltd.).Subsequently, after a week of recovery period, the rats were dividedinto four groups (Control group, Compound A-1 group, Compound A-2 group,and Compound A-3 group; n=17 each) shown in Table 1 by stratifiedrandomization assignment using body weight as an index, and test dietswere dietary administered to them. At the 15th day from the start, ofdietary administration, the light/dark cycle was advanced by 8 hours,and after that, dietary administration of test diets was continued for 2weeks. The light/dark cycle of the breeding environment excluding thephase advance date was set to 12 hours cycle, and the illuminance of thelight period was set to be approximately 200 lx (185-230 lx). In thismodel of mild sleep disorder with the phase advance, the influence oftest feed administration on the light period activity level after thephase advance was examined.

The test was carried out by a two-sided test, the significance level ofthe test was set to 5%, and the trend difference was 10% on both sides.For the analysis, SAS software release 9.3 (manufactured by SASinstitute Japan Ltd.) was used.

TABLE 1 Group (n = 17) Test diet Control MF Compound A-1 MF containing0.0005% of Compound A (Equivalent to 0.62 mg/kg BW/day) Compound A-2 MFcontaining 0.005% of Compound A (Equivalent to 6.25 mg/kg BW/day)Compound A-3 MF containing 0.05% of Compound A (Equivalent to 62.46mg/kg BW/day) MF: powder feed for raising mice and rats (MF)(manufactured by Oriental Yeast Co., Ltd.). Compound A:(3S,8aS)-3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Compound 1as above) BW: Body Weight

[Results]

FIG. 4 shows the transition of the light period activity of rats afterthe phase advance. The horizontal axis represents the number of dayselapsed since phase advance, the vertical axis represents the activitylevel in the light period (12 hours), and the activity level shows themean value±standard error. For pre 3 days, the mean value of the lightperiod activity level during the 3-day pre-administration period wasshown.

From the shape of the graphs, it is shown that the activity level risesafter the phase advance, and returns to the activity level before thephase advance with the passage of days.

As shown in FIG. 4, there was a particularly noticeable rise in theactivity level in the light period on Day 1 and Day 2. In order toconfirm the effect of Compound A on the activity level of Day 1 and Day2 during the light period, Dunnett test with mixed model for repeatedmeasures method was carried out. The Compound A-3 group showed atendency to suppress the activity level in compared with the Controlgroup (P=0.0978). Compound A-2 group significantly inhibited theactivity level in compared with the Control group (P=0.0481). From thesefacts, it was suggested that Compound A suppresses sleep disturbance byphase advance during the light period from Day 1 to Day 2.

FIG. 5 shows the activity level of rats on Day 1, light period (12hours) after the phase advance. The horizontal axis represents eachgroup, the vertical axis represents the activity level in the lightperiod (12 hours), and the activity level was the average value±standarderror.

A group comparison was done by Dunnett test. As a result, the Compound.A-3 group tended to suppress the activity level in compared with theControl group (P=0.0536), and the Compound A-2 group significantlysuppressed the activity level in compared with the Control group(P=0.016). From these facts, it was suggested that Compound A suppressesdeterioration of sleep due to phase advance in the light period (12hours) on Day 1.

As described above, both FIGS. 4 and 5 show significant difference afterthe phase advance in the early light period where the activity level ismost likely to be disturbed. Therefore, it was suggested that Compound Aimproves sleep quality.

(Example 3) Method Described in Wada T. et al., Brain Res Bull. 2006;69: 388-92

An electrode for EEG is implanted in the brain of an 8-week-old maleWistar rat, and a recovery period is set for 1 week after surgery. Afterthat, habituation to brush stimulation (sleep deprivation stimulus) isperformed for 2-3 days. On test days, while monitoring EEG data, ifnon-REM sleep is detected, the rat is stimulated with a brush to preventnon-REM sleep. The effect of test drug on the influence of prevention ofNon-REM sleep is examined.

(Example 4) Method Described in Miyazaki K. et al., PLoS One. 2013; 8:e55452

Male C3H—HeN mice aged 8 to 15 weeks old are subjected to stress bychanging breeding on paper flooring in a cage with a rotating basket tothat in 1.5 cm water-immersed state at room temperature on Day 1 of thetest. In this state, the mice become to live on a rotating basket inorder to avoid water immersion. The sleep/wakefulness of mice areevaluated by monitoring the activity status of the mice with themovement of the rotating basket. If evaluated after 7 days as a load,the evaluation in EEG and the evaluation by activity level will besimilar.

(Example 5) Method Described in Sei H. et al., Life Sci. 2003; 73: 53-59

Male Wistar rats aged 10 to 12 weeks old are raised for 1 to 7 daysafter the change of the light/dark cycle of 12-hour dark period/12-hourlight period to the light/dark cycle of 4-hour dark period/12-hour lightperiod. For these animals, it is possible to assess the body temperaturerhythm by installing a telemetry type thermometer in the abdominalcavity in advance. It is also possible to detect the modulation of bodytemperature rhythm and to assess the influence on plasma cortisolconcentration and hippocampal BNDF protein mass, caused by changing thelight/dark cycle.

Sequence Listing Free Text

The base sequence represented by SEQ ID NO: 1 is a base sequence ofHSP70 forward primer.

The base sequence represented by SEQ ID NO: 2 is a base sequence ofHSP70 reverse primer.

The base sequence represented by SEQ ID NO: 3 is a base sequence of beta2 microglobulin forward primer.

The base sequence represented by SEQ ID NO: 4 is a base sequence of beta2 microglobulin reverse primer.

1. A heat shock protein expression-inducing agent comprising a compoundrepresented by formula (I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.2. The heat shock protein expression-inducing agent according to claim1, wherein R is isopropyl, phenyl, or hydroxyphenyl.
 3. The heat shockprotein expression-inducing agent according to claim 1, wherein R isisopropyl.
 4. The heat shock protein expression-inducing agent accordingto claim 1, which is in the form of a pharmaceutical composition,quasi-drug composition or food and beverage composition.
 5. A sleepimproving agent comprising a compound represented by formula (I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.6. The sleep improving agent according to claim 5, wherein R isisopropyl, phenyl, or hydroxyphenyl.
 7. The sleep improving agentaccording to claim 5, wherein R is isopropyl.
 8. The sleep improvingagent according to claim 5, which is in the form of a pharmaceuticalcomposition, quasi-drug composition or food and beverage composition. 9.An autonomic nerve-adjusting agent comprising a compound represented byformula (I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.10. The autonomic nerve-adjusting agent according to claim 9, wherein Ris isopropyl, phenyl, or hydroxyphenyl.
 11. The autonomicnerve-adjusting agent according to claim 9, wherein R is isopropyl. 12.The autonomic nerve-adjusting agent according to claim 9, which is inthe form of a pharmaceutical composition, quasi-drug composition or foodand beverage composition.
 13. An anti-stress agent comprising a compoundrepresented by formula (I):

wherein R is a lower alkyl, phenyl, or hydroxyphenyl, or a salt thereof.14. The anti-stress agent according to claim 13, wherein R is isopropyl,phenyl, or hydroxyphenyl.
 15. The anti-stress agent according to claim13, wherein R is isopropyl.
 16. The anti-stress agent according to claim13, which is in the form of a pharmaceutical composition, quasi-drugcomposition or food and beverage composition.